Biomedical subjects

E Ninio

Publications and source records attributed to E Ninio.

At least 37 records · Page 2Linked to original sources

PubMed1996

Pefabloc, 4-[2-aminoethyl]benzenesulfonyl fluoride, is a new, potent nontoxic and irreversible inhibitor of PAF-degrading acetylhydrolase.

We report here that 4-[2-aminoethyl]benzenesulfonyl fluoride (Pefabloc SC, Pefabloc), a new irreversible serine proteinase inhibitor, efficiently inhibits both human and rat platelet activating factor (PAF)-degrading acetylhydrolase (acetylhydrolase). Indeed, low concentrations of Pefabloc (0.1 mM) rapidly and totally inactivate both human plasma-, VLDL-, IDL-, LDL- and HDL-associated acetylhydrolase, and in addition, acetylhydrolase synthesized and released by human adherent monocytes in culture, as well as rat brain cytosolic acetylhydrolase. By contrast, Pefabloc only minimally inhibited the phospholipase A2 (PLA2) activity from Naja naja and from porcine pancreas. In addition, Pefabloc is relatively nontoxic, stable and convenient to use. Henceforth, Pefabloc may replace both DFP and PMSF and therefore constitutes a useful and valuable tool in future studies of acetylhydrolase.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Phagocytic activation induces formation of platelet-activating factor in human monocyte-derived macrophages and in macrophage-derived foam cells. Relevance to the inflammatory reaction in atherogenesis.

Monocyte-derived macrophages and macrophage-derived foam cells in arterial tissue may undergo phagocytic activation and thereby contribute to an inflammatory reaction. We have investigated the effect of phagocytic activation on the formation of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF-acether, PAF), a proinflammatory phospholipid, in human monocyte-derived macrophages (macrophages) and in cholesterol-loaded macrophage foam cells (foam cells). Adherent human monocyte-derived macrophages were transformed into foam cells upon incubation with acetylated low-density lipoproteins (Ac-LDL). Such foam cells characteristically displayed a markedly increased content of cholesteryl esters compared with macrophages (4.3 +/- 1.3 microgram/microgram DNA and 0.2 +/- 0.3 microgram/microgram DNA, n = 5, respectively). After phagocytic stimulation with serum-opsonized zymosan (OPZ), both macrophages and foam cells synthesized PAF transiently with maximal production (0.5-1.1 pmol PAF/microgram DNA, n = 5, corresponding to 4.0-8.8 pmol PAF/10(6) cells, as assessed by bioassay) occurring approximately 15 min after stimulation. A major fraction of the synthesized PAF remained cell-associated; such PAF was composed mainly of the hexadecyl (16:0 PAF, approximately 75%) and the octadecenyl (18:1 PAF) species and of trace amounts of octadecyl (18:0 PAF), as assessed by reverse-phase liquid chromatography. Addition of exogenous 16:0 lyso-PAF alone triggered PAF formation (0.9-1.7 pmol PAF/microgram DNA, after 15 min of cellular stimulation); simultaneous cellular stimulation with OPZ and 16:0 lyso-PAF increased PAF formation in an additive manner. Acetyltransferase, the enzyme which acetylates the precursor lyso-PAF and transforms it into PAF, displayed elevated activity both in macrophages and in foam cells, attaining 83-240 pmol PAF formed per min per mg DNA (n = 4); such elevated activity was not increased by OPZ-stimulation. The activity of acetylhydrolase, the PAF-degrading enzyme, was similar in macrophages and in foam cells, and varied between 120 pmol and 320 pmol PAF degraded per min per mg DNA (n = 5). Cell-associated acetylhydrolase activity was increased significantly by 40+/-15 % (P < 0.003, n = 5) after 15 - 30 min of activation with OPZ compared with non-stimulated cells and may account for the rapid decrease in cellular PAF content observed approximately 30 min after stimulation. These studies have established that metabolism of PAF in foam cells closely resembles that in macrophages, and thus PAF metabolism is largely independent of cellular cholesterol content. Moreover our data are consistent with the hypothesis that both macrophages and macrophage-derived foam cells upon phagocytic-activation constitute a significant transient source of PAF at inflammatory sites in the arterial intima where this phospholipidic mediator may exert potent proatherogenic and prothrombotic effects.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1996

Identification on human CD36 of a domain (155-183) implicated in binding oxidized low-density lipoproteins (Ox-LDL).

Uptake of oxidized LDL (oxLDL) by macrophages is one of the key events implicated in the initiation and perpetuation of atherosclerotic lesions. One of the major scavenging receptors, which binds modified LDL, on macrophages is CD36. The domain on CD36 implicated in the binding of oxLDL remains to be elucidated. In this study, COS cells transfected with human CD36 cDNA bound FITC-oxidized human LDL in a dose-dependent, saturable manner. This binding was inhibited by an excess of oxLDL but not by native LDL. Anti-CD36 monoclonal antibodies (mAbs) 10/5, FA6-152, and 8A6 (directed against domain 155-183), but not mAb 13/10 (directed against domain 30-76), completely inhibited oxLDL binding to human CD36-transfected COS cells. Cells transfected with a chimeric human CD36 construct (hmh 155-183), resulting from the swapping of human domain 155-183 with its murine counterpart, resulted in low binding of oxLDL. In contrast, cells transfected with a chimeric murine CD36 construct (mhm 155-183), resulting from the swapping of murine domain 155-183 with its human counterpart, resulted in high binding of oxidized human LDL. Binding of oxLDL to cells transfected by chimeric construct mhm 155-183 were only partially blocked by mAbs 10/5, FA6-152, and 8A6. In the present study we have identified, for the first time, an important functional domain (encompassing amino acids 155-183) on CD36 involved in the binding of oxLDL. In addition, the binding site for oxidized human LDL on murine CD36 seems to differ from its human counterpart.

Animals↗

PubMed1995

PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme.

In human plasma, platelet activating factor (PAF)-degrading acetylhydrolase (acetylhydrolase) is principally transported in association with LDLs and HDLs; this enzyme hydrolyzes PAF and short-chain forms of oxidized phosphatidylcholine, transforming them into lyso-PAF and lysophosphatidylcholine, respectively. We have examined the distribution, catalytic characteristics, and transfer of acetylhydrolase activity among plasma lipoprotein subspecies separated by isopycnic density gradient ultracentrifugation; the possibility that the plasma enzyme may be partially derived from adherent monocytes has also been evaluated. In normolipidemic subjects with Lp(a) levels < 0.1 mg/mL, acetylhydrolase was associated preferentially with small, dense LDL particles (LDL-5; d = 1.050 to 1.063 g/mL) and with the very-high-density lipoprotein-1 subfraction (VHDL-1; d = 1.156 to 1.179 g/mL), representing 23.9 +/- 1.7% and 20.6 +/- 3.2%, respectively, of total plasma activity. The apparent Km values for PAF of the enzyme associated with such lipoproteins were 89.7 +/- 23.4 and 34.8 +/- 4.5 mumol/L for LDL-5 and VHDL-1, respectively: indeed, the Km value for LDL-5 was some 10-fold higher than that of the light LDL-1, LDL-2, and LDL-3 subspecies, whereas the Km of VHDL-1 was some twofold greater than those of the HDL-2 and HDL-3 subspecies. Furthermore, when expressed on the basis of unit plasma volume, the Vmax of the acetylhydrolase associated with LDL-5 was some 150-fold greater than that in LDL-1 (d = 1.019 to 1.023 g/mL). No significant differences in the pH dependence of enzyme activity or in sensitivity to protease inactivation, sulfydryl reagents, the serine protease inhibitor Pefabloc, or the PAF antagonist CV 3988 could be detected between apo B-containing and apo A-I-containing lipoprotein particle subspecies. Incubation of LDL-1 (Km = 8.4 +/- 2.6 mumol/L) and LDL-2 (d = 1.023 to 1.029 g/mL; Km = 8.4 +/- 3.3 mumol/L) subspecies with LDL-5, in which acetylhydrolase had been inactivated by pretreatment with Pefabloc, demonstrated preferential transfer of acetylhydrolase to LDL-5. Acetylhydrolase transferred to LDL-5 from the light LDL subspecies exhibited a Km of 9.4 +/- 2.2 mumol/L, a value characteristic of the particle donors. Finally, acetylhydrolase (Km = 23.4 +/- 7.6 mumol/L) released by adherent human monocytes in culture was found to bind preferentially to small, dense LDL subspecies upon incubation of Pefabloc-inactivated plasma with monocyte supernatant.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗

PubMed1994

PAF-acether-degrading acetylhydrolase in plasma LDL is inactivated by copper- and cell-mediated oxidation.

In peripheral blood, native low-density lipoprotein (LDL) is a major carrier of acetylhydrolase, the enzyme that hydrolyzes the sn-2 acetate of PAF-acether, converting it to lyso PAF-acether. By controlling the level of PAF-acether, the acetylhydrolase may regulate the biologic effects of this potent inflammatory and thrombotic mediator. The biologic oxidation of LDL appears to underlie its atherogenicity. We report here that oxidative modification of LDL led to progressive loss of associated acetylhydrolase activity. Reductions of approximately 90% and 40% of acetylhydrolase activity occurred respectively in LDL oxidized for 24 hours by copper ions (2.5 mumol/L) in phosphate-buffered saline and in LDL incubated with human monocyte-like THP1 cells in Ham's F-10 medium. Acetylhydrolase activity decreased as a function of the degree of LDL oxidation and was correlated with an increase in net negative charge and in the content of thiobarbituric acid-reactive substances (r = -.94 and r = -.88, respectively; P < or = .001). The acetylhydrolase of mildly oxidized LDL displayed a similar Km for PAF-acether compared with native LDL, whereas its Vmax was lower. Thus, acetylhydrolase conserved its affinity for PAF-acether, whereas a nondefined and noncompetitive inhibitor, apparently produced during oxidation, might account for the observed loss in enzymatic activity. Acetylhydrolase activity was totally recovered in LDL modified by both acetylation and malondialdehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1994

Effects of organometals on cellular signaling. II. Inhibition of reincorporation of free arachidonic acid and influence on paf-acether synthesis by triethyllead.

Organometal compounds affect many enzymes, especially those containing SH-groups as acyl- and acetyltransferases involved in lysophospholipid reacylation. In HL-60 cells, organotin and -lead compounds stimulate phospholipase A2 activity, contributing thus to increase the level of lysophospholipids. In the present study, we have tested whether paf-acether (paf) biosynthesis was affected by treatment with triethyllead (Et3PbCl) in HL-60 cells. Et3PbCl inhibits the incorporation of exogenous arachidonic acid in the presence of high (> or = 50 microM) but not low concentrations (< or = 1 microM). High concentrations of the lead compound are unable to induce paf formation by itself, however, lower concentrations (< or = 10 microM) acted synergistically with TPA or fMLP to stimulate paf formation. Whereas unstimulated cells produced 0.4 pmole paf/2 x 10(6) cells, the stimulation with low fMLP (0.1 microM) resulted in the synthesis of 1.7 pmole and with low TPA (2 ng/ml) in 0.5 pmole paf. Preincubation of the cells with 10 microM Et3PbCl for 20 to 30 min increased the amount of paf formed by these cells to 3.3 pmole after treatment with 0.1 microM fMLP and 1.5 pmole after TPA. Furthermore, the results showed an inhibition of acetyltransferase (the key enzyme of paf synthesis) by the high and not by low concentrations of the lead compound. We conclude that low concentrations of Et3PbCl (< or = 10 microM) may act as a synergistic inducer of paf synthesis initiated via a receptor-coupled stimulation.

Acetyl-CoA C-Acetyltransferase↗

PubMed1993

Biosynthesis of platelet-activating factor in cultured mast cells. Involvement of the CoA-independent transacylase demonstrated by analysis of the molecular species of platelet-activating factor.

We have recently demonstrated that arachidonate [20:4(5,8,11,14)] was primarily linked to the hexadecyl (16:0) and octadecenyl (18:1) species of alkylacyl derivatives of glycerolphosphocholine (GroPCho). Consistent with the involvement of arachidonate-specific CoA-independent transacylase in the synthesis of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-GroPCho), 16:0 and 18:1 PAF species were formed upon antigen stimulation [Joly, F., Breton, M., Wolf, C., Ninio, E. & Colard, O. (1992) Biochim. Biophys. Acta 1125, 305-312]. In the present work, addition of lyso-PAF to mast cells resulted in PAF production. We analyzed the PAF species formed in the presence of a defined lyso-PAF molecular species in order to differentiate between either direct acetylation or involvement of the membrane precursor. The 18:1 lyso-PAF was more effective than the 16:0 in producing PAF which was composed of 95% 18:1 PAF, the balance being 16:0, indicating that part of the acetylated lyso-PAF originated from the cellular pool of alkyl-arachidonyl-GroPCho in resting cells. Consistent with alkyl-arachidonyl-GroPCho species content and acetyltransferase specificity, similar amounts of 16:0 and 18:1 PAF species were formed when mast cells were stimulated with antigen. Supplemented with 16:0 or 18:1 lyso-PAF, antigen-stimulated mast cells responded by 230% and 125% increase in PAF synthesis, respectively. As expected, the amount of the PAF species corresponding to the added lyso-PAF was increased. More interestingly, addition of 16:0 lyso-PAF almost doubled the amount of 18:1 PAF content as compared to antigen alone, thus indicating that the lyso-PAF formed via the CoA-independent transacylase was significantly used for PAF synthesis, despite a large excess of exogenous lyso-PAF. The CoA-independent transacylase, measured using [3H]lyso-PAF as a substrate in sonicates from antigen-stimulated cells, was decreased concurrently with PAF formation. In conclusion, we show that when lyso-PAF is added to mast cells, a direct acetylation may occur. However, PAF is preferentially synthesized through a mechanism involving the CoA-independent transacylase reaction.

Acyltransferases↗

PubMed1993

Autocrine amplification of PAF-acether formation in immunologically activated murine macrophages.

When murine macrophages activated in vivo with bacille Calmette-Guérin were triggered with either acetyl-CoA or propionyl-CoA to form PAF-acether (PAF), similar amounts of platelet-aggregating product were recovered. Liquid chromatographic purification and reversed-phase analysis showed that the composition of PAF molecular species formed in the presence of acetyl-CoA was an equimolar mixture of PAF bearing C16:0 alkyl chain (57% +/- 7, mean +/- SD, n = 3) and PAF C18:1. The PAF-like material obtained from the propionyl-CoA-supplemented macrophages was a mixture of the propionyl analogue of PAF (66% +/- 11, n = 3) and native PAF. The rate of lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) reaction in a macrophage lysate was similar for either substrate in the presence of an equimolar mixture of propionyl-CoA and acetyl-CoA. We conclude that the exogenously added propionyl-CoA is transferred to lyso-PAF acceptor to form propionyl-PAF by the PAF-forming acetyltransferase. Propionyl-PAF triggers the formation of native PAF probably from the endogenous acetyl-CoA pool. Two specific PAF antagonists, BN 52021 (60 microM) and WEB 2086 (3 microM), did not influence the rate of PAF synthesis in the presence of either acetyl-CoA or propionyl-CoA and did not prevent native PAF formation when propionyl-CoA was added alone, suggesting that the classical PAF receptors are not involved. This is the first description of a possible mechanism of autocrine amplification of PAF biosynthesis in macrophages.

Acetyl Coenzyme A↗

PubMed1993

Human platelets release a paf-acether: acetylhydrolase similar to that in plasma.

Intact washed human platelets aggregated in response to paf-acether (paf) and did not metabolize [3H]paf at concentrations up to 10 nM. However, when platelets were lysed by exposure to pH 9.5, resulting in 37.5 +/- 2.5% (mean +/- SD, n = 3) lactic dehydrogenase (LDH) release, 20.5 +/- 5.7% of the radioactivity was detected as labeled lyso paf and 5.7 +/- 3.1% as labeled alkylacylglycerophosphocholine. When platelets were aggregated with 0.5 IU/mL thrombin or high concentrations of paf (100 nM), they released a part of their acetylhydrolase without releasing LDH. In supernatants obtained from aggregated platelets, 21 +/- 2% or 10 +/- 2% (n = 3), respectively, of the total platelet acetylhydrolase activity was detected vs. none in supernatants of resting cells. The release of acetylhydrolase was concentration- and time-dependent and paralleled the release of PF 4, a marker for alpha-granules. The acetylhydrolase affinity for paf (Km) measured in sonicates of resting and thrombin-activated platelets was 8.3 +/- 1.5 microM vs. 10.6 +/- 1.5 microM, n = 5, n.s. in a "Mann Whitney" test. The latter Km was slightly but significantly different (P < 0.05, n = 5) from that of the thrombin-released acetylhydrolase (7.9 +/- 1.5 microM) and that of the latter was itself different from plasma acetylhydrolase (5.3 +/- 0.5, P < 0.05, n = 5). Addition of plasma (acid-treated to inactivate acetylhydrolase) decreased the Km value of supernatant acetylhydrolase to 6.1 +/- 1.4 microM. All preparations of acetylhydrolase exhibited similar pH requirements and sensitivity to various inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1992

Biosynthesis of paf-acether in cultured-mouse mast cells: the role of calcium and G proteins.

We examined the potential role of a guanine nucleotide-binding protein in the biosynthesis of paf-acether (paf) and the release of beta-hexosaminidase during antigenic stimulation of cultured mouse bone marrow-derived mast cells. Unlike pertussis toxin, cholera toxin treatment enhanced the antigen-stimulated production of paf and calcium mobilisation without affecting acetyltransferase activation and cell degranulation. The level of intracellular cAMP doubled in cholera toxin-treated cells. Our data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the IgE receptor-mediated signal transduction leading to paf production most probably at the level of Ca2+ influx.

Acetyltransferases↗

PubMed1992

Heterogeneity of arachidonate and paf-acether precursor pools in mast cells.

In mammalian cells, arachidonate release and paf-acether formation are frequently associated. The alkyl-acyl-GPC has been proposed as an important source for released arachidonic acid and arachidonate-containing alkylacyl-GPC species as unique precursor for paf-acether. However, the specificity of precursor pools either concerning arachidonic acid or paf-acether is still a matter of controversy. We studied the relationship between the precursor pools for both autacoids in antigenically-stimulated cultured mast cells. We took advantage of the particular arachidonate turnover rate in each phospholipid to investigate the role of alkyl-arachidonyl-GPC in the supply of arachidonic acid by using newly and previously [14C]arachidonate-labeled cells. The specific activity of the released arachidonate was reduced 2-fold following overnight cell incubation, whereas labeling in alkyl-arachidonoyl-GPC was only slightly modified and never corresponded to that of released arachidonate when newly or previously labeled cells were triggered with the antigen. These results are not in favor of a major role for alkyl-arachidonoyl-GPC in supplying arachidonate. In contrast, by using previously labeled cells, we demonstrated that all arachidonate-containing phospholipids were involved in the release of arachidonic acid. The pattern of alkyl chains in alkyl-arachidonoyl-GPC, as well as in total alkylacyl-GPC, is unique since it consists mainly of 18:1 (more than 55%), whereas the 16:0 represents only about 30% of total alkyl chains. Therefore, we analyzed paf-acether molecular composition in order to compare it to the alkyl composition of the precursor pools. The content in 18:1 species of paf-acether, as measured by bioassay (aggregation of rabbit platelets), was always lower than that of 16:0 species and then did not correspond to the alkyl composition of the precursor. These data suggest that the enzymes involved in paf synthesis might be specific for 16:0 alkyl chains of precursor pool.

Acetyltransferases↗

PubMed1991

Biosynthesis of paf-acether. XVII. Regulation by the CoA-independent transacylase in human neutrophils.

Treatment of intact human polymorphonuclear neutrophils (PMN) with low concentrations of phorbol myristate acetate (PMA, 1-10 ng/ml) induced paf-acether (paf) and lyso paf formation, arachidonate release, and simultaneous inhibition of CoA-independent lyso paf: transacylase as assayed in a cell-free system. Inhibition of [3H]lyso paf reacylation was also observed when it was exogenously added to the PMA-treated intact PMN. When higher concentrations of PMA (40-100 ng/ml) were used, paf biosynthesis was severely impaired and the level of the CoA-independent transacylase activity returned to basal level. Since lyso paf appears to be the substrate for PMA-activated paf formation (remodeling pathway), we showed that [14C]acetate was incorporated into the paf molecule. By contrast, labeling with [3H]choline was not appropriate in this model. The presented results are against the involvement of a de novo route in paf synthesis initiated by PMA and open a new possibility of an important role for the CoA-independent transacylase in controlling the level of lyso paf availability for paf formation.

Acetates↗

PubMed1991

Biosynthesis of paf-acether. XVI. Acetyltransferase specificity determines composition of paf-acether molecular species in human polymorphonuclear neutrophils.

In human neutrophils, the velocity of the lyso paf-acether:acetyl-CoA acetyltransferase reaction was almost 2-fold higher in the presence of lyso paf-acether bearing a 16:0 alkyl chain at the sn-1 position of glycerol than in that of its 18:0 analog. The paf-acether produced from an equimolar mixture of the two substrates was a 5:1 mixture, respectively, of the 16:0 and 18:0 species. The ratio of 16:0/18:0 lyso paf-acether in microsomal fractions, as analyzed by gas chromatography, was close to 1, whereas the paf-acether formed in these fractions from endogenous phospholipids was nearly exclusively of the 16:0 form. We conclude that acetyltransferase possesses a higher affinity for 16:0 than for 18:0 lyso-PAF and thus might control the molecular composition of paf-acether synthesized by stimulated human polymorphonuclear neutrophils.

Acetyl-CoA C-Acetyltransferase↗

PubMed1991

Kinetic studies of human and rat neutrophil lysoPAF acetyltransferase using lysoPAF and dansyllysoPAF as substrates.

Enzyme kinetic studies of lysoPAF acetyltransferase from microsomal preparations of human and rat neutrophils were carried out using lysoPAF or dansyllysoPAF as substrate. With the human enzyme, incomplete conversion of the substrate into the product was observed at 37 degrees C with both substrates. The acetyltransferase was inactivated at 37 degrees C in the absence of substrate with a half-life of 7.5 min. However, the initial rate of product formation under the assay conditions was linear up to 10 min. Both enzymes were optimally active at 40 microM concentration with either substrate, but enzyme activity was inhibited at higher substrate levels. At a constant substrate concentration (40 microM), the Km (microM) and Vmax (nmol product/min/mg protein) values for the human acetyltransferase, with respect to acetyl-CoA were 132 and 23.1, respectively, with lysoPAF as substrate, and 105 and 26.7, respectively, when dansyllysoPAF was used. The Km and Vmax values for the rat enzyme were 105 and 6.5, respectively, with lysoPAF as substrate, and 120 and 5.4, respectively, when dansyllysoPAF was used. Under our standard conditions, lysoPAF required 1 mg of BSA per mL in the assay, whereas full activity of both enzymes was seen with dansyllysoPAF even in the absence of BSA. The results show that dansyllysoPAF can replace lysoPAF in the assay without any significant changes in kinetic parameters.

Acetyl Coenzyme A↗

PubMed1991

Transmembrane signalling and paf-acether biosynthesis.

Expression of lyso paf-acether (lyso paf):acetyl-CoA acetyltransferase and its activation above basal levels by specific agonists controls the rate of paf biosynthesis in proinflammatory cells. Acetyltransferase activation in these cells is due to the rapid postranslational modification of an inactive precursor by phosphorylation, most probably catalyzed by a cAMP-dependent kinase. However, the possibility exists that a calcium/calmodulin-dependent kinase can be implicated as well. Unlike murine cultured mast cells, human neutrophils form paf when stimulated with phorbol myristate acetate (PMA) or diacylglycerol. In both cell types, acetyltransferase is activated by PMA. Controversy exists as to whether PMA activates the remodeling pathway, i.e. the activation of phospholipase A2 and acetyltransferase, or the de novo route through CDPcholine cholinephosphotransferase action on alkylacetylglycerol. There is some indication that PKC might regulate paf biosynthesis. The implication of a GTP-regulated protein has also been postulated in signal transduction leading to paf formation in endothelial cells, neutrophils, and mast cells. The topography of paf formation is discussed in light of the subcellular distribution of acetyltransferase in neutrophils and Krebs II cells.

Acetyltransferases↗

PubMed1991

Cell adhesion by membrane-bound paf-acether.

It was recently shown that paf-acether (paf) synthesized in different cell types remains partly cell-associated. In the present work, we tested the hypothesis that cell-associated paf might in fact remain exposed on the external plasma membrane and be able to exert its biological functions. Human polymorphonuclear neutrophils (PMN), stimulated with either opsonized zymosan or ionophore A23187 and then thoroughly washed, induced aggregation of human and rabbit platelets in a time- and dose-dependent manner, whereas no aggregation was observed in the presence of unstimulated cells. Aggregation was inhibited by the specific paf antagonists BN 52021 or WEB 2086. Treatment of stimulated PMN with specific anti-paf antibody before addition to platelets abolished the PMN--paf-mediated aggregation. Microscopic observation of human platelets revealed that aggregates formed by platelets were attached to the neutrophil surface. Paf remained associated with PMN following human PMN-human platelet interaction, in contrast to human PMN-rabbit platelet incubation, where it disappeared from both PMN and platelet surfaces. Our results strongly support the hypothesis that a fraction of cell-associated paf synthesized in neutrophils is located on and/or in the external plasma membrane, where it can act upon other cells by direct cellular contact. Such a mechanism of cell adhesion might play a role in cell physiology (neutrophils but also monocytes/macrophages, eosinophils, and lymphocytes), as well as in the onset and perpetuation of immune and inflammatory reactions.

Animals↗

PubMed1990

Biosynthesis of paf-acether. Activators of protein kinase C stimulate cultured mast cell acetyltransferase without stimulating paf-acether synthesis.

Antigen stimulation of cultured bone-marrow-derived mast cells sensitized with specific monoclonal IgE induced cell degranulation and paf-acether (paf; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) biosynthesis via the deacylation/acetylation (remodelling) pathway. Phorbol myristate acetate (PMA; 20-100 ng/ml) triggered only acetyltransferase activation, without concomitant lyso-paf (1-O-alkyl-sn-glycero-3-phosphocholine) and paf formation. A low concentration of PMA (5 ng/ml) potentiated antigen-induced degranulation, acetyltransferase activation and paf formation by about 30% but did not change the level of lyso-paf formation. Stimulation of mast cells with antigen increased intracellular Ca2+ from 61 to 269 nM, whereas no modification of Ca2+ influx was observed when cells were pretreated with PMA (5 ng/ml) before antigen challenge. Gas chromatography coupled to electron capture detection revealed that the composition of paf formed by cells stimulated by antigen alone was similar to that of paf formed by PMA-primed antigen-stimulated cells; 84 +/- 8% and 79 +/- 2% (means +/- S.E.M., n = 3) of molecules respectively bore the C16:0 alkyl chain moiety, with the remainder bearing essentially C18:0 molecules. Overnight treatment of mast cells with PMA (200 ng/ml) caused disappearance of protein kinase C (PKC) from both cytosol and membranes. When such cells were stimulated further with antigen, they failed to degranulate, and acetyltransferase activation, paf production and lyso-paf production were decreased by 33 +/- 11%, 57 +/- 4% and 96 +/- 3% respectively (n = 3 or 5). The PKC inhibitors chlorpromazine and staurosporine inhibited to a significant extent both cell degranulation and all steps leading to paf biosynthesis. Our data suggest that PKC-dependent mechanisms are operational during cell degranulation and contribute only in part to paf biosynthesis. The PKC-dependent signal directly generated by PMA or diacylglycerol is not sufficient to trigger the full cell response, which is obtained only through receptor-operated antigen challenge.

Acetyltransferases↗

PubMed1990

Paf-acether-induced superoxide anion generation in human B cell line.

Paf-acether (paf) and lyso phospholipids induced an oxydative burst on EBV-transformed B lymphocyte cell line. Superoxide anion formation measured by lucigenin-dependent chemiluminescence was dependent on both paf concentration and time-course of challenge. Paf C18:0 at 10 microM was more potent than its C16:0 analogue at the same concentration. Choline-containing phospholipids with 2-acyl (long chain) were inactive. The paf antagonists BN 52021 and WEB 2086 structurally unrelated to paf were inactive whereas paf structural analogue CV 3988 inhibited superoxide formation induced by paf and lysophospholipids. Such a phospholipid-induced oxydative burst in B cells might exert an effect in the numerous pathophysiological situations where large amounts of paf are produced by phagocytic cells.

B-Lymphocytes↗